Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are members of the neurotrophin family which can improve neuronal survival and prevent neurodegeneration resulting from disease or trauma. Sensory, sympathetic and cholinergic neurons are sensitive to NGF. Therapy using NGF is assumed to prevent the development of Alzheimer's disease, to prevent the loss of brain function in stroke and to improve the patient's quality of life in peripheral diabetic neuropathy. In addition, NGF is suggested to be useful for the treatment of neuronal damage and as a targeting agent for neuroectodermal tumors. For reviews on the use of NGF in therapy, see Saragovi H. U. and Burgess K., Expert Opinion in Therapeutic Patents 1999, 9:737-751; Schulte-Herbruggen O. et al., Curr. Med. Chem. 2007, 14(22): 2318-2329.
In the CNS, BDNF acts as a potent trophic factor for cerebral and spinal motor neurons that degenerate in amyotrophic lateral sclerosis (Thoenen H. et al., C. R. Acad. Sci. III, 1993, 316(9): 1158-63), and for dopaminergic neurons of substantia nigra that are lost in Parkinson's disease (Spina M. B. et al., J. Neurochem. 1992, 59(1):99-106). In the periphery, BDNF has a neurotrophic activity for small fibrous neurons involved in a number of sensory neuropaties (Lindsay R. M., Neurobiol. Aging. 1994, 15(2):249-251).
The biological effects of NGF, BDNF and other neurotrophins are mediated by the binding to cellular receptors of two classes: high affinity (Kd of 10−11) receptors of the tyrosine kinase family and a low affinity (Kd of 10−9) p75 receptor. The p75 receptor is a glycoprotein with a molecular weight of 75 kDa. It has no intrinsic catalytic activity, but is associated with the ERK family of soluble kinases (Volonte C. et al., Mol. Biol. Cell 1993, 4(1):71-78) and has a role in the protection of neurons against apoptosis. All the neurotrophins can bind to this receptor.
The specificity of individual neurotrophins is determined by their binding to p140trk, a particular type of tyrosine kinase (Trk) receptors, with NGF and NT-3 binding to TrkA, while BDNF and NT-4/5 binding to TrkB. The binding is followed by the receptor dimerization, resulting in autophosphorylation of intracellular tyrosine residues of the receptor by internal domains of the kinase. This in turn initiates a cascade of enzymatic reactions that mediate biological effects of neurotrophins, including an increased survival of neurons (Barbacid M., J. Neurobiol. 1994, 25(11): 1386-1403).
Neurotrophins are homodimers composed of two identical subunits each having approximately 120 amino acid residues and bound by hydrophobic interactions. X-ray structure analysis conducted for the NGF homodimer (McDonald N. Q. et al., J. Mol. Biol. 1991, 219(4): 595-601) and for the BDNF/NT-3 heterodimer (Robinson R. C. et al., Biochemistry 1995, 34(13): 4139-46) demonstrated a common 3D structure of neurotrophins. They all contain exposed structural units called loops 1, 2, 3 and 4 which are hairpin structures three of which have beta-turn sections at their ends (the so-called sections A-A″, A′″-B and C-D, loops 1, 2, 4) and one section of 3 consecutive reverse turns (called section B-C, loop 3). These hairpin structures are thought to be responsible for the specific binding to a particular type of neurotrophin receptors.
Using site-directed mutagenesis (by transplanting loop 2 from BDNF into NGF), the specific binding of BDNF to TrkB receptor was shown to be determined by loop 2. This chimeric neurotrophin could bind to TrkB rather than TrkA and displayed a BDNF-like activity (Ibanez C. F. et al., EMBO J. 1993, 12(6): 2281-93). Furthermore, additional residues of loops 3 (Gln84) and 4 (Lys96 and Arg97) are important for the TrkB activation, but are thought not to be involved in its binding. All these residues are located on the surface of the molecule.
For NGF, it was shown that a stretch of amino acids from 29 to 35 corresponding to the loop 2 is responsible for the specific binding to TrkA. Synthetic peptides corresponding to the NGF sequence 29-35 were found to be NGF antagonists. Loop 1 was responsible for the binding to p75 receptor (McDonald and Chao. Structural determinants of neurotrophin action. JBC 270, 19669-19672, 1995; Longo et al. Synthetic NGF peptides prevent neuronal death via p75 receptor-dependent mechanism. J. Neurosci. Res., 1997).
The ability of neurotrophins to protect neurons in experimental models of neurodegenerative disease gave rise to the optimism concerning their potential therapeutic applications. In fact, however, neurotrophins were unsuccessful drugs because, being proteins, they are unavailable orally, unable to cross the blood-brain barrier and other biological barriers and are rapidly degraded in the bloodstream. Moreover, neurotrophins have pleiotropic actions and can induce carcinogenesis. The interaction of neurotrophins with the p75 receptor induces neuronal death by apoptosis. A considerable drawback of neurotrophins is the development of pain syndrome upon their use. This is related to the involvement of neurotrophins in the endogenous regulation of pain. Clinical applications of neurotrophins were unsuccessful (Penn R. D. et al., Neurosurgery 1997, 40(1):94-99; discussion 99-100, 1997). This led to attempts at producing their low molecular weight analogs, stable in the bloodstream and capable of crossing the gastro-intestinal and blood-brain barriers. It was suggested that low molecular weight analogs of neurotrophins would produce only partial biological effects due to their presumably selective interaction with some of the binding sites on a subpopulation of the neurotrophin receptors and thus would only activate the desired units of signaling cascades. There are several patents for low molecular weight peptide analogs of neurotrophins. Linear peptides having sequences that correspond to the respective sequences of hairpin structures exhibited an antagonist activity (Longo F. M. et al., Cell Regul. 1990, 1(2): 189-195). Peptide agonists of neurotrophins were obtained from among cyclic peptides able to maintain a conformation similar to the beta-turn conformation of neurotrophin loops. The provision of an agonist activity required the creation of bivalent analogs mimicking the homodimer structure of neurotrophins. Thus, Longo et al. (F. M. Longo et al., 1999, U.S. Pat. No. 5,958,875) have patented cyclic monomeric and dimeric peptides having sequences that correspond to a.a. 43-47 and 92-97 of NGF (loops 2 and 4). The cycles are formed using disulfide bonds at accessory cysteine or penicillamine residues introduced into said peptides. When tested in vitro, bicyclic analogs demonstrated an agonist activity.
Similar compounds have been patented by Saragovi et al. (H. U. Saragovi et al., 2002, U.S. Pat. No. 6,017,878). Cyclic peptide analogs are formed using disulfide or other (ionic, metal chelate etc.) bonds between linear peptides with sequences that fully or partially correspond to a.a. 28-36 of loop 1, 42-49 of loop 2, 59-67 and 91-99 of loops 3 and 4 in NGF. A minimal molecular weight of a cyclic peptide having an agonist activity is 1500 dalton, the peptide comprising 16 amino acids. The peptides are active in vitro.
Richard Hughes (R. A. Hughes et al., 2000, PCT WO 00/75176 A1) has filed a patent application for mono-, bi- and tricyclic peptide analogs of loops 2 and 4 in BDNF having both agonist and antagonist activities. Disulfide bridges are used for the cyclisation, and the cycles are linked both via the disulfide bridges and through carboxyl and amine groups of the side chain amino acids. The spacer length affects the activity, with maximal activity displayed by bivalent analogs with the same distance between the cyclic peptide fragments as in BDNF. An agonist activity is also displayed by monomeric cyclic peptides, analogs of the p75 receptor binding site at loop 4 of BDNF.
Thus, in all cases the binding to neurotrophin receptors is provided by the presence of an extended site that generally corresponds to a pentapeptide (except for a tripeptide site at loop 4 of BDNF that interacts with the p75 receptor), and the formation of an active beta-turn conformation requires that this site is cycled. In the presence of the Trk receptor binding, an agonist activity is achieved by combining two of these cycles into a bivalent entity.
The compounds described in the references cited above, however, neither disclose nor suggest any novel structural variations of the claimed compounds.